ATR (Attenuated Total Reflectance) spectroscopy is a technique that is based on molecular vibration and the curvature of light beams when passing through different mediums. An ATR spectrum is generated by transmitting radiation, which can be IR (from about 0.1 cm to about 7.5×10−5 cm), VIS (from about 7.0×10−5 to about 4.0×10−5 cm), or UV (from about 4.0×10−5 cm to about 2.2×10−5 cm), through an optical crystal in contact with a sample and then determining what portion of the incident radiation is attenuated by the sample at a particular wavelength. ATR spectrometry is used extensively in clinical assays, medical diagnostics, and laboratory testing. Since the depth of penetration for the evanescent wave in ATR spectrometry is shallow, there is a low incidence of Fresnel Reflection. Thus, reliable spectral analysis of murky, semisolid, turbid, and optically dense solutions is possible with ATR spectrometry.
When light travels from one medium to another, a speed change results that causes the light to bend. The amount that a beam of light bends on passing from a first medium to a second medium can be determined by calculating the refractive index of both mediums, defined as the ratio of the speed of light in a vacuum to the speed of light in a medium (n=c/v), and applying Snell's Law. Snell's Law: n1 sin θ1=n2 sin θ2 (where n1 is the refractive index of the first medium, n2 is the refractive index of the second medium, sin θ1 is the angle of light to the normal in the first medium, and sin θ2 is the refracted angle of the light to the normal in the second medium), calculates the amount of curvature of the beam of light on moving from the first medium to the second medium. Pursuant to Snell's Law, when the beam of light impinges an interface between the first and second medium at or above a critical angle, defined as sincrit (or θcrit)=sin−1 n2/n1there is no refracted ray, i.e., the incident light is totally internally reflected, and an evanescent wave is generated.
In ATR spectrometry, a sample is measured by passing a beam of light through an optical crystal, which can be mounted on a probe. The beam, which, for example, can be UV, IR, or VIS, is directed onto the optical crystal at an angle of incidence such that all incident light undergoes total internal reflection. When the beam undergoes total internal reflection, an electro-magnetic radiation field, described by N. J. Harrick (1965) as an evanescent wave, extends beyond the surface of the crystal into the sample next to the crystal. The depth of penetration of the evanescent wave, which is generally quite shallow, is a function of the refractive index of the crystal material, refractive index of the sample material, angle of incidence of the beam, and wavelength of the light. In regions of the spectrum where the sample absorbs energy, the evanescent wave is attenuated and the attenuated energy is passed back to the beam of light. The beam of light then exits the optical crystal and impinges a detector. The detector records the attenuated beam, which can then be transformed to generate a spectrum, e.g., an absorption spectra.
Detectors used in spectroscopy generally fall into two classes, photographic detectors, in which radiation impinges upon an unexposed photographic film, and electronic detectors, in which the radiation impinges upon a detector and is converted into an electrical signal. Electronic detectors provide the advantage of increased speed and accuracy, as well as the ability to convert the spectral data into an electronic format, which can be displayed, processed, and/or stored. Examples of electronic detectors include photomultiplier tubes and photodetectors. Photomultiplier tubes are quite sensitive, but are relatively large and expensive. Photodetectors provide the advantage of reduced size and cost. Some examples of photodetectors are pin diode detectors, charge coupled device detectors, and charge injection device detectors.
According to the Beer-Lambert Law, a linear relationship exists between the spectrum and the concentration of a sample. In mathematical terms: A=εbc, where A is the absorbance value of a sample at a specific wavelength, b is the pathlength through the sample, c is the concentration, and ε is the absorbency coefficient of the material at the specific wavelength. In order to determine the relationship between the spectrum and the concentration, an instrument measures a set of standard samples, which reflect the compositions of unknown samples as closely as possible and span the expected range of concentrations and compositions of the unknowns. The measurements of the standard samples along with measured data from a training set are then used to create a set of calibration equations. However, in order to apply the equations to a set of unknown samples, finding the constant for the absorptivity coefficient is necessary. As the absorptivity coefficient for a given compound at a selected wavelength is constant, a least squares regression method, classical least squares regression method, or inverse least squares regression method can be used to solve the equation. Once the calibration equations have been solved, calculation of quantities or properties of unknown samples is possible. However, in order for the quantities and properties to be predicted accurately, the unknown samples should be measured under the same conditions. Spectrometers are ideal measurement devices because unlike other methods, which give single point measurements for each calibration and unknown sample, the spectrum of a sample contains many data points. Furthermore, every response value in a spectrum has some relation to the properties or constituents that make up the sample.
Dissolution testing is required for all solid oral pharmaceutical dosage forms in which absorption of the drug is necessary for the product to exert the desired therapeutic effect. One way to calculate the amount of dissolution of a substance in a medium is by creating and solving a calibration equation that accurately predicts the quantity of the constituents of interest.
The U.S. Pharmacopoeia (USP) is one well-known standard source of information that provides for dissolution and drug release testing in the majority of monographs for such dosage forms. Exceptions are for tablets meeting a requirement for completeness of solution or for rapid (10 to 15 minutes) disintegration of soluble or radiolabled drugs. The apparatus and procedure conform to the requirements and specifications given, e.g., USP 23rd edition Chapter 711 (Dissolution) pages 1791-1793. Dissolution testing serves as a measure of quality control, stability and uniformity as well as a means by which to correlate in-vitro with in-vivo drug release characteristics. Current USP dissolution methods most commonly employ a temperature programmable water bath, maintained at about 37° C., in which sample vessels are submerged. These vessels contain a predetermined volume of a dissolution media and a mechanism to agitate the contents of the vessel. This may be accomplished with a rotating basket attached to a shaft or with a paddle that is also attached to a shaft, both of which are generally described in USP 23rd edition Chapter 711 (Dissolution) pages 1791-1793. The solid dosage form is placed into the media-filled vessel at time zero, and specific vessel temperature and mixing speeds are maintained as dissolution of the dosage form in the medium is monitored over time.
A number of systems are currently used to perform dissolution testing of dosage forms. For example, it is known to use a pumping system which removes dissolution media from the vessel and then provides it to a spectrometer for analysis. However, this system has the disadvantage of removing the dissolution media from the vessel during dissolution, thereby, changing the dissolution conditions. It is also known to use fiber optic flow cell probes disposed within the dissolution media to monitor dissolution. However, such probes have apertures which may become clogged, thus, affecting the dissolution measurements.